Mesothelin: a New Target for Immunotherapy

Vol. 10, 3937–3942, June 15, 2004 Clinical Cancer Research 3937

Perspective Mesothelin: A New Target for Immunotherapy

Raffit Hassan, Tapan Bera, and Ira Pastan ISOLATION OF MAB K1 AND CLONING OF ITS Laboratory of Molecular Biology, National Cancer Institute, NIH, 40-KDA ANTIGEN, MESOTHELIN Bethesda, Maryland The Mab K1 was generated by immunization of mice with the human ovarian carcinoma cell line OVCAR-3 (3). The ABSTRACT reactivity of Mab K1 against a variety of different human tumor Mesothelin is a differentiation antigen present on nor- cell lines was tested using immunofluorescence (3). It showed mal mesothelial cells and overexpressed in several human reactivity with several ovarian cancer cell lines, including tumors, including mesothelioma and ovarian and pancreatic OVCAR-2, OVCAR-3, OVCAR-5, A-1847, and SKOV3; cer- adenocarcinoma. The mesothelin gene encodes a precursor vical cancer cell lines HeLa and KB; and gastric cell lines AGS protein that is processed to yield the 40-kDa protein, me- and HTB103. No reactivity was detected with breast, colon, or sothelin, attached to the cell membrane by a glycosylphos- prostate cancer cell lines. Care must be taken with the use of phatidyl inositol linkage and a 31-kDa shed fragment named Mab K1 because its reactivity is acid labile (5). megakaryocyte-potentiating factor. The biological function The reactivity of Mab K1 against normal human tissues of mesothelin is not known. Mesothelin is a promising can- was tested by immunohistochemistry using cryostat tissue sec- didate for tumor-specific therapy, given its limited expres- tions (3). Most normal tissues showed no reactivity with Mab sion in normal tissues and high expression in several can- K1, with the exception of mesothelial cells that line the perito- cers. SS1(dsFv)PE38 is a recombinant anti-mesothelin neal, pleural, and pericardial cavities. There was also weak immunotoxin that is undergoing clinical evaluation in pa- reactivity with the basal cells of the trachea and cells in the tients with mesothelin-expressing tumors. There is evidence Fallopian tubes. A similar immunoreactivity was seen in cryo- that mesothelin is an immunogenic protein and could be stat tissue sections of cynomolgus monkeys, making monkeys a exploited as a therapeutic cancer vaccine. A soluble me- useful model for preclinical toxicology studies. Although mesothelin variant has been identified and could be a useful sothelin RNA has been found in extracts of normal lung, kidney, tumor marker for malignant mesotheliomas. and liver, its presence is due to the fact that these tissues contain a mesothelial cell lining. Our data as well as data from other INTRODUCTION investigators indicate that there is no mesothelin expression in Mesothelin is a 40-kDa cell surface glycoprotein that is the parenchymal cells of these organs (3, 6). highly expressed in pancreatic cancers, ovarian cancers, me- The antigen recognized by Mab K1 was identified by sotheliomas, and some other cancers (1). Mesothelin is not a expression cloning using cDNA prepared from a HeLa cell line. cancer-specific antigen, but like CD20, it is a differentiation The gene was named mesothelin because immunohistochemical antigen that is present on normal cells and highly expressed in studies showed that the membrane-bound antigen it encodes is many cancers. Mesothelin is expressed on normal mesothelial present on normal mesothelial cells (1). Alignment of the cDNA cells lining the pleura, pericardium, and peritoneum. The limited sequence with the human genome sequence shows that the distribution of mesothelin on normal tissues makes it a promis- mesothelin gene has 17 exons on human chromosome 16p13.3 ing target for tumor-specific therapy. Also, because small (Fig. 2). The mesothelin cDNA is 2138-bp long, contains an amounts of mesothelin can be detected in the blood of some open reading frame of 1884 bp, and encodes a 69-kDa protein. patients with mesothelin-positive cancers, measurement of me- Two minor spliced forms of the major mesothelin transcript sothelin in the blood may be useful for the diagnosis and to have been detected that encode two slightly altered proteins follow the course of some of these patients (2). The mesothelin termed variant 1 and variant 2 (Fig. 2). Variant 1 has an gene encodes a 69-kDa precursor protein that is processed to a insertion of 8 amino acids after glutamine 408. This variant was 40-kDa membrane-bound protein termed mesothelin and a 31- present in the original cDNA clone isolated from HeLa cells (1). kDa shed fragment called megakaryocyte-potentiating factor Variant 2 retains the intron between exons 16 and 17 and (MPF) that is released from the cell (Fig. 1). The mesothelin probably leads to premature termination of the protein, resulting protein is the antigen recognized by the monoclonal antibody in its release from the cell. There are now over 60 mesothelin (Mab) K1, whereas MPF was isolated from the medium of a sequences in the expressed sequence tag database. Only two of human pancreatic cancer cell line (3, 4). these contain the insertion at position 408, and four others encode the prematurely terminated protein. The major cell-associated glycoprotein recognized by Mab K1 has a molecular mass of ϳ40-kDa, although some full- length 71-kDa glycosylated protein has also been detected (1). Received 12/23/03; revised 3/16/04; accepted 3/24/04. The precursor protein contains 628 amino acids and has four Grant support: A Career Development Award from the American potential N-linked glycosylation sites. A furin cleavage site, Society of Clinical Oncology (R. Hassan). RPRFRR, is present at amino acids 288–293. Mesothelin is Requests for reprints: Raffit Hassan, Laboratory of Molecular Biol- ogy, National Cancer Institute, 37 Convent Drive, Room 5116, Be- made as a 69-kDa polypeptide with a hydrophobic sequence at thesda, MD 20892-4264. Phone: (301) 451-8742; Fax: (301) 402-9469; the carboxyl end that is removed and replaced by phosphatidy- E-mail: [email protected]. linositol. After glycosylation at one or more of its four putative Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2004 American Association for Cancer Research. 3938 Mesothelin: A New Target for Immunotherapy

Fig. 1 Schematics showing maturation of mesothelin protein. Precursor protein for mesothelin is synthesized as a 622- amino acid polypeptide with a calculated molecular mass of 77 kDa. The potential signal peptide (SP) and the glycosylphosphatidyl inositol anchor signal sequence (GASS) are predicted

at the NH2 terminus and the COOH terminus, respectively. The precursor protein has four predicted glycosylation sites (CHO) and a furin cleavage site (RR). Cleavage at the furin site generates membrane-bound mesothelin (green) and the secre- tory protein megakaryocyte-potentiating factor (red).

glycosylation sites, it is cleaved by furin to yield the 40-kDa highest activity, and MPF was purified from the conditioned fragment that was first found on the surface of OVCAR-3 cells medium of these cells. On SDS-PAGE analysis, MPF was and a smaller 32-kDa fragment that is released from the cell. As identified as a single band with a molecular mass of approxi- described above, this 32-kDa fragment is MPF, which was mately 32-kDa. Glycopeptidase F digestion and amino sugar initially isolated from the medium of the human pancreatic analysis demonstrated that MPF is a glycoprotein containing at cancer cell line HPY-5 (4). least one N-linked sugar chain. Using a megakaryocyte colony- forming assay, the purified MPF potentiated megakaryocyte MPF colony formation in the presence of interleukin-3. However, MPF is a 32-kDa protein that stimulates the megakaryocyte MPF alone did not have any intrinsic megakaryocyte colony- colony-forming activity of murine interleukin-3 in mouse bone stimulating activity. Whether MPF has megakarocyte-potentiat- marrow cell culture (4). Yamaguchi et al. (4) examined 64 cell ing activity in humans is unknown, but it is of interest that lines for their ability to produce megakaryocyte-potentiating patients with mesotheliomas often have elevated platelet counts. activity. The pancreatic cancer cell line HPC-Y5 showed the Subsequently, the cDNA encoding human MPF was iso-

Fig. 2 Schematics describing the mesothelin gene and the proteins it encodes. A, genomic organization of mesothelin gene. There are 17 exons (filled boxes) in the mesothelin gene. B, drawing of three different forms of mesothelin protein described in the database. Mesothelin is the most common variant and is predicted to be membrane-bound; mesothelin V-1 has an additional 8 amino acids (yellow) and is also predicted to be bound to the membrane. Mesothelin V-2 has a modified COOH terminus (orange), lacks the glycosylphosphatidyl inositol anchor signal sequence, and is predicted to be secreted.

lated and found to encode a polypeptide consisting of 622 amino gested that mesothelin might have a role in adhesion because acids with a deduced molecular mass of 68 kDa, although the 3T3 cells transfected with a mesothelin expression vector were human MPF secreted by HPC-Y5 cells is only 32 kDa in size more difficult to remove from the culture dishes than nontrans- (7). The cDNA encoding MPF is identical to the mesothelin fected cells (1). The possibility that mesothelin may play a role cDNA. Both the mesothelin and MPF cDNA encode the same in adhesion is supported by a recent study showing that me- precursor protein, which has a furin cleavage site. Cleavage by sothelin binds to CA125 and that this interaction mediates cell furin leads to a shed 32-kDa protein called MPF, and a 40-kDa adhesion. Based on these findings, the authors (9) suggested that fragment that remains attached to the cell membrane by a there may be an important role for CA125 and mesothelin in the glycosylphosphatidyl inositol linkage is called mesothelin (Fig. metastatic spread of ovarian cancer. 1). The gene encoding this precursor protein is referred to as Several studies have generated data indicating that activa- Mesothelin, MPF, or Mesothelin/MPF gene. tion of specific signaling pathways that are important in cancer can lead to an increase in mesothelin expression. Yamashita et SOLUBLE MESOTHELIN al. (10) reported that in the Eker rat carcinoma cell line, there is increased expression of the Erc gene, the rat homolog of the A soluble 42–45-kDa protein with an NH -terminal amino 2 human mesothelin gene. Eker rats develop hereditary renal acid sequence, identical to that of the membrane-bound portion carcinomas due to mutations of the tumor suppressor gene Tsc2. of mesothelin, was identified using the murine Mab OV569, ␤-Catenin levels are also elevated in Eker rat tumors (11). The obtained by immunizing mice with malignant ascites of a patient mesothelin gene is also differentially regulated by members of with ovarian cancer (2). The antigen to which OV569 binds was the Wnt signal transduction pathway (12). Using C57MG mouse also detected in the cell-free culture supernatant from antigen- mammary epithelial cells, mesothelin was up-regulated by positive carcinomas and in the cell-free malignant effusions of Wnt-1. Interestingly, tumors with constitutive activation of the patients with mesothelin-positive carcinomas. The gene se- Wnt signaling pathway, such as ovarian and pancreatic cancers, quence and analysis of the expressed sequence tag database of have high mesothelin expression. Additional studies are needed mesothelin indicate that the major form of mesothelin is at- to fully define mesothelin function as well as the role of me- tached to the cell surface and is not soluble. The soluble form of sothelin in carcinogenesis. mesothelin is likely due to an abnormal splicing event resulting in a frameshift mutation and premature termination at amino acid 600 deleting the amino acids at the COOH terminus that are MESOTHELIN EXPRESSION IN HUMAN responsible for its association with the cell membrane. Prema- MALIGNANCIES ture termination is probably due to lack of splicing of the intron Mesothelin gene expression in different human cancers has between exons 16 and 17 (Fig. 2). Only 4 of the 60 expressed been studied using serial analysis of gene expression (SAGE) sequence tags in the database, which align to the 3Ј end of the tag analysis. The presence and frequency of a specific tag for a mesothelin gene, could produce this soluble form of mesothelin, particular gene are indicative of the frequency of expression of but it is also possible that soluble mesothelin is a proteolytically that gene in different tissues or cancers (13). The presence of cleaved fragment of membrane-bound mesothelin. mesothelin protein has been studied by immunohistochemistry To test whether soluble mesothelin-related proteins (SMR) using anti-mesothelin Mabs K1 and 5B2. The commercially could be valuable for diagnosis or follow-up of cancer patients, available anti-mesothelin Mab 5B2 (Novocastra, Newcastle-on- Scholler et al. (2) examined sera from 68 healthy donors, 3 Tyne, United Kingdom) is an IgG1 murine Mab that was ob- patients with inflammatory disease, 1 patient with a benign tained by immunizing mice with a recombinant prokaryotic tumor, and 105 patients with different tumors. Serum from all fusion protein corresponding to approximately 100 amino acids patients without cancer was negative for this soluble antigen. that are present in the membrane-bound form of the mesothelin However, sera from 23 of 30 (77%) patients with ovarian cancer molecule. were antigen positive. This antigen was also detected in the The only normal tissues that show strong mesothelin im- serum of some patients with other tumors such as breast and munoreactivity are the mesothelial cells of pleura, pericardium, lung cancer. When validated, this test may be useful for the and peritoneum. On immunohistochemistry, a strong staining of diagnosis and follow-up of patients with mesothelin-expressing a single layer of mesothelial cells is seen (3). Mesothelin- malignancies (see below). Because this soluble form of me- positive cancers exhibit a diffuse membranous staining of the sothelin is present in very small amounts in the blood, it should tumor cells, usually of similar intensity to that seen in normal not interfere with antibody-based therapies that target the me- mesothelial cells. The percentage of tumor cells that are mesothelin antigen on cancer cells. sothelin positive varies with specimen quality, with better results seen with frozen tissue, especially when using Mab K1. BIOLOGICAL FUNCTION OF MESOTHELIN Mesothelin Expression in Ovarian Cancer. The immu- To study the biological function of mesothelin in vivo,we nohistochemical localization of Mab K1 in ovarian cancer was generated mutant mice in which both copies of the mesothelin initially tested using cryostat tissue sections (3). Ten of the 15 gene were inactivated (8). To our surprise, there were no de- (66%) nonmucinous ovarian cancers examined were mesothelin tectable abnormalities in the mutant mice in terms of growth and positive, whereas none of the 4 mucinous ovarian cancers were reproduction as compared with their wild-type littermates. Also, positive. A study performed on paraffin-embedded tissue sec- there was no statistical difference in platelet counts between tions using the anti-mesothelin antibody 5B2 also showed me- wild-type and mesothelin knockout mice. It was originally sug- sothelin immunopositivity in the majority of cases of nonmuci-

nous ovarian cancer (6). Other studies have noted high apies. In addition, biodistribution studies of the anti-mesothelin mesothelin expression at both the mRNA and protein levels in Mab K1 in nude mice show that it localizes to mesothelin- samples from serous ovarian carcinoma (14). There are many expressing tumors and could therefore be useful for imaging or tags for the mesothelin gene present in nine SAGE libraries immunotherapy of such tumors (5). made from ovarian cancers, supporting the fact that mesothelin Development of the Anti-Mesothelin Immunotoxin, is overexpressed in ovarian cancer. SS1(dsFv)PE38 (SS1P). Because the murine anti-mesothelin Mesothelin Expression in Mesotheliomas. Using cryo- Mab K1 by itself is not cytotoxic toward mesothelin-positive stat sections obtained from patients with pleural mesothelioma, cells, we decided to develop anti-mesothelin immunotoxins Mab K1 reactivity was observed in all 15 cases of epithelial using the potent bacterial toxin Pseudomonas exotoxin A (PE). mesotheliomas tested, but in none of the 4 cases of sarcomatous This toxin consists of three functional domains: (a) a cell- mesothelioma tested (15). Similar results, showing mesothelin binding domain that causes binding to a cell surface protein; (b) positivity in epithelial mesotheliomas, were seen in a recent a translocation domain that brings the active fragment of the study (16) evaluating mesothelin expression in paraffin-embed- toxin to the cytosol; and (c) an ADP-ribosylation domain that ded tissue sections of mesotheliomas, using the anti-mesothelin inhibits protein synthesis leading to cell death (21). PE38 is a antibody 5B2. SAGE tag analysis shows that there are 55 tags truncated 38-kDa form of PE that is not toxic to target cells for mesothelin cDNA in the single library made from a perito- because the cell binding domain is deleted (22). Conjugation of neal mesothelioma. PE38 to an antibody results in targeting of the toxin to the Mesothelin Expression in Pancreatic Cancer. In the antigen recognized by the antibody and produces cell death. study reported by Argani et al. (17), the tag for mesothelin was The first anti-mesothelin immunotoxin consisted of Mab consistently present in pancreatic cancer samples, but not in K1 chemically linked to a modified form of PE38 (23). Al- normal pancreas. Mesothelin mRNA was confirmed to be pres- though it had antitumor activity, it had drawbacks for clinical ent in the cancer cells by in situ hybridization in 4 of 4 resected use including its low affinity and large size, which limits tumor primary pancreatic adenocarcinomas and by immunohistochem- penetration. Therefore, efforts were directed toward developing istry using the 5B2 antibody in all 60 resected primary pancre- a molecule with favorable clinical properties leading to the atic adenocarcinomas. Labeling was intense (Ն3ϩ)in54ofthe development of SS1(dsFv)PE38 (SS1P). SS(scFv) is an anti- 60 samples tested. No mesothelin reactivity was noted in the mesothelin single chain Fv (scFv) that was obtained from the adjacent normal pancreas. splenic mRNA of mice using antibody phage display (24). Other studies have also confirmed mesothelin expression in Using “hot spot” mutagenesis, the affinity of SS(scFv) was the majority of pancreaticobiliary tumors (6, 18). Currently improved over 15-fold to yield SS1(scFv) (25). Because immu- there are 19 tags for mesothelin in four different SAGE libraries notoxins containing a scFv are often unstable at 37°C, we have made from pancreatic cancers. developed a method of stabilizing the Fv by replacing the Mesothelin Expression in Other Tumors. Mesothelin peptide linker connecting the light and heavy chains with a immunoreactivity was noted in the majority of frozen tissue sec- disulfide bond (26). The SS1scFv was converted to a disulfide tions from squamous cell carcinomas of the cervix, head and neck, stabilized Fv (dsFv) and used to construct the recombinant vulva, lung, and esophagus (19). However, this immunoreactivity immunotoxin SS1(dsFv)PE38. This immunotoxin has signifi- in squamous cell carcinomas was much less when formalin-fixed, cant antitumor activity in vitro and in vivo against mesothelin- paraffin-embedded tissue sections were examined (6, 20). Other positive tumor cells and was chosen for evaluation in clinical tumors that show some mesothelin expression include lung adeno- trials and given the name SS1P. carcinomas, endometrial carcinoma, biphasic synovial sarcomas, Activity of SS1P against Mesothelin-Expressing Tu- and desmoplastic small round cell tumors (6, 20). mors in Nude Mice. The antitumor activity of SS1P was Very little or no expression of mesothelin is seen in breast evaluated using both a tumor xenograft and metastatic model. adenocarcinomas, carcinomas of thyroid, renal cell carcinoma, A431-K5 (human epidermoid carcinoma cell line expressing transitional cell carcinoma of the bladder, melanomas, and hep- mesothelin by transfection) cells were established as s.c. tumors atomas (6). Mesothelin expression by immunohistochemistry is in athymic nude mice. Animals with growing tumors received also infrequently seen in tumors of the gastrointestinal tract. In three i.v. injections of SS1P on days 5, 7, and 9 after the the case of gastric adenocarcinoma, mesothelin expression is injection of tumor cells. Mice treated with 4, 6, or 8 ␮g/dose of seen in 14–29% of cases (3, 6). This low frequency of mesothe- SS1P had complete tumor regression.1 The activity of SS1P in lin positivity in gastric tumors by immunohistochemistry is inhibiting lung metastases was also evaluated. Mice received i.v. somewhat surprising because SAGE tag analysis shows that injection with either mesothelin-positive tumor cell line NCI- mesothelin is highly expressed in many gastric cancer speci- H226 (derived from a pleural mesothelioma) or mesothelin- mens. There are currently 151 SAGE tags for the mesothelin negative lung adenocarcinoma cell line PC14PE6. When SS1P gene in six SAGE tag libraries made from gastric cancer tissues. was administered to these nude mice, SS1P selectively inhibited the growth of lung metastases produced by the mesothelin- producing NCI-H226 cells (27). These data indicate that SS1P MESOTHELIN AS A TARGET FOR TUMOR- can be active in a metastatic setting. SPECIFIC IMMUNOTHERAPY Given the high mesothelin expression in several human malignancies and its limited expression in normal tissues, mesothelin is a good target for tumor-specific antibody-based ther- 1 Unpublished data.

Activity of SS1P against Mesothelin-Expressing Human SOLUBLE MESOTHELIN AS A DIAGNOSTIC Tumor Cells. The cytotoxic activity of SS1P against tumor TUMOR MARKER cells obtained from patients was evaluated in vitro. The activity The small amount of mesothelin shed into the serum could of SS1P against human ovarian cancer cells obtained from make it a valuable diagnostic tool in cancers that express me- patients undergoing cytoreductive surgery was evaluated using a sothelin. Using a double determinant (sandwich) ELISA, the three-dimensional in vitro culture system. Using tumor cells serum concentrations of SMR were assayed in samples obtained obtained from five patients with ovarian cancer and one with from 44 patients with mesothelioma, 160 patients with other cervical squamous cell carcinoma and apoptosis as an end point, inflammatory or malignant lung and pleural diseases, and 68 we showed that tumors expressing mesothelin showed a signif- matched healthy controls, of whom 40 had asbestos exposure icant dose-dependent sensitivity to SS1P with cytotoxic activity (32). Thirty-seven of the 44 patients (84%) with mesothelioma observed at concentrations as low as 1 ng/ml. No antitumor had elevated SMR, in contrast to only 3 of 160 patients (2%) activity was seen at 100 ng/ml in tumors that did not express with other diseases of the lung and pleura. In patients with mesothelin (28). Similarly, the activity of SS1P against tumor mesothelioma, SMR correlated with the stage and tumor burden, cell lines established from ascites of patients with peritoneal with elevated SMR levels in patients with advanced stage and mesotheliomas was evaluated using a cell proliferation assay. Seven tumor cell lines were established from the ascites of increased disease burden. No correlation between SMR and patients with peritoneal mesothelioma. Six of the seven cell platelet counts in patients with mesothelioma was noted. In the lines expressed mesothelin, whereas one cell line did not. Cell healthy control group of 68 patients, 28 patients who had no lines that expressed mesothelin were very sensitive to SS1P, asbestos exposure had no elevation in SMR, whereas 7 of the 40 patients with exposure to asbestos had increased serum SMR. with IC50 values ranging between 0.08 and 3.9 ng/ml. The peritoneal mesothelioma cell line that did not express mesothe- Within 1–5 years, three of these seven patients developed me- Ͼ 2 sothelioma, and one developed lung carcinoma. This study lin was resistant to SS1P with an IC50 of 100 ng/ml. suggests that serum SMR could be a helpful tumor marker for the diagnosis of mesothelioma. It could also potentially be CLINICAL TRIALS OF SS1(DSFV)-PE38 (SS1P) helpful in screening asbestos-exposed individuals for early ev- Based on the preclinical studies showing antitumor activity idence of developing mesothelioma. of SS1P against mesothelin-expressing tumors, as well as tox- The value of soluble mesothelin for the diagnosis of ovarian icology studies in monkeys, SS1P has been approved for inves- cancer is also being studied. It appears that soluble mesothelin by tigational use in patients with mesothelin-positive tumors. Two itself lacks sensitivity and specificity as a tumor marker in ovarian institutional review board-approved Phase I studies of SS1P are cancer but may complement CA125 for the detection of ovarian currently ongoing (29, 30). One study involves giving SS1P as cancer (33). Additional studies of the utility of mesothelin, in an i.v. bolus injection every other day for three or six doses. The combination with other ovarian tumor markers, are needed to fully other involves administration of SS1P as a continuous i.v. define its role as a screening test for ovarian cancer. infusion over 10 days. Both studies are open to accrual, and dose escalation is ongoing. MESOTHELIN IMMUNOHISTOCHEMISTRY MESOTHELIN AS A THERAPEUTIC CANCER FOR TUMOR DIAGNOSIS VACCINE Initial studies using cryostat sections of human tumors There is experimental evidence to suggest that mesothelin suggested that Mab K1 could help distinguish epithelial me- is a strongly immunogenic protein. In a Phase I clinical trial of sothelioma from lung adenocarcinoma because lung adenocar- patients with pancreatic cancer who were vaccinated with irra- cinomas were uniformly mesothelin negative (15). However, diated, granulocyte macrophage colony-stimulating factor-se- two recent studies using the anti-mesothelin Mab 5B2 showed creting pancreatic tumor cell lines, a dose-dependent systemic mesothelin positivity in 41–53% of lung adenocarcinomas (6, antitumor immunity against autologous tumors was seen (31). 20). Despite differences in the pattern of mesothelin staining in Some patients treated in this study had clinical benefits that may epithelial mesotheliomas (in which the staining is membranous) have led to an improvement in progression-free and overall and lung adenocarcinomas (in which the staining is predomi- survival. All patients who had this benefit had a strong anti- nately cytoplasmic), it is unlikely that mesothelin immuno- mesothelin T-cell immune response as measured by the ELIS- staining will help the pathologist to distinguish between these POT assay. Based on these results, preclinical studies to develop two tumors. However, because mesothelin immunostaining is a a therapeutic mesothelin vaccine for the treatment of mesothe- very sensitive positive marker for epithelial mesothelioma, a lin-expressing cancers are ongoing.3 negative mesothelin immunostain suggests a diagnosis other than mesothelioma (16). Another tumor type in which mesothelin immunostaining may be of value to the pathologist is pancreatic adenocarcinoma because mesothelin expression is noted only in the cancerous 2 L. Quian, C. F. Verschraegen, J. Mendoza, and R. Hassan: Cytotoxic tissue and not in normal pancreas or other benign histologies, activity of the recombinant anti-mesothelin immunotoxin, SS1(dsFv) PE38, towards tumor cell lines established from ascites of patients with such as chronic pancreatitis (6, 17, 18). This is especially peritoneal mesotheliomas. Anticancer Research, in press. important in the interpretation of pancreatic fine-needle aspira- 3 Dr. Elizabeth Jaffe, personal communication. tion specimens, a procedure now commonly used for initial

pathological diagnosis, given the cytologic overlap between 15. Chang K, Pai LH, Pass H, et al. Monoclonal antibody K1 reacts malignant and reactive processes (34). with epithelial mesothelioma but not with lung adenocarcinoma. Am J Surg Pathol 1992;16:259–68. CONCLUSION 16. Ordo´n˜ez NG. Value of mesothelin immunostaining in the diagnosis of mesothelioma. Mod Pathol 2003;16:192–7. Mesothelin is a differentiation antigen that is highly ex- 17. Argani P, Iacobuzio-Donahue C, Ryu B, et al. Mesothelin is over- pressed in several malignancies and is being evaluated as a expressed in the vast majority of ductal adenocarcinomas of the pan- target for antibody- and vaccine-based therapies of cancer. Also, creas: identification of a new pancreatic cancer marker by serial analysis soluble mesothelin could be valuable as a tumor marker for of gene expression (SAGE). Clin Cancer Res 2001;7:3862–8. diagnosis and follow-up of patients with mesothelin-expressing 18. Hassan R, Laszik Z, Lerner MR, et al. Mesothelin, a cell surface malignancies. In addition, immunohistochemistry studies eval- glycoprotein, as a target for tumor specific therapy of pancreatic cancer. uating mesothelin expression show it to be valuable to the Proc Am Soc Clin Oncol 2003;22:283. pathologist for tumor diagnosis. 19. Chang K, Pastan I, Willingham M. Frequent expression of the tumor antigen CAK1 in squamous-cell carcinomas. Int J Cancer 1992; 51:548–54. ACKNOWLEDGMENTS 20. Miettinen M, Sarlomo-Rikala M. Expression of calretinin, throm- We thank Drs. Claire Verschraegen (Health Science Center, Univer- bomodulin, keratin 5, and mesothelin in lung carcinomas of different sity of New Mexico, Albuquerque, NM), Elizabeth Jaffee (The Sidney types: an immunohistochemical analysis of 596 tumors in comparison Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD), with epithelioid mesotheliomas of the pleura. Am J Surg Pathol 2003; Bruce W. S. 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Raffit Hassan, Tapan Bera and Ira Pastan

Clin Cancer Res 2004;10:3937-3942.

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